Background: Recent interest in naturally based products has increased. Various herbal extracts are known to have a variety of medicinal properties. Among the various natural medicines, safflower seeds have beneficial effects on various bone diseases such as bone fracture, osteoporosis, and osteodysplasia. In addition, they are known to have anti-inflammatory effects. The objective of this study was to evaluate the effect of a safflower seed extract (SSE) on the regeneration of periodontal tissue in a preclinical 1-wall model in dogs. Methods: Preclinical 1-wall periodontal defects were surgically created in the mesial aspect of the maxillary third and mandibular fourth premolar and in the distal aspect of the maxillary first and mandibular second premolar, and were randomly assigned to receive SSE/collagen (SSE/Col), phosphate-buffered saline/collagen (buffer control), or root planing only (surgical control). The created 1-wall defect configuration was 4 mm in depth by 4 mm in width. We selected the segment showing the best activity to the osteoblast cells that was sensitive to the formation of calcified nodules among the SSE fractions extracted from various organic solvents. The animals were euthanized at 8 weeks postsurgery, and block sections of the defects were collected for histologic and histometric analysis. Results: The junctional epithelium migration did not show any statistically significant differences among the treatments. In connective tissue adhesion, the SSE/Col group and the buffer control group showed significant differences compared to the surgical control group. New cementum averaged 3.84 ± 0.57 mm, 3.75 ± 0.24 mm, and 1.53 ± 1.22 mm for the SSE/Col group, the buffer control group, and the surgical control group, respectively, with the SSE/Col and buffer control groups significantly different from the surgical control group (P <0.05). The amount of intrabony cementum in the SSE/Col group was significantly different (P <0.01) from the surgical control group, but the amount of suprabony cementum did not demonstrate any statistical difference between the different treatments. The amount of new alveolar bone averaged 2.93 ± 0.70 mm, 2.10 ± 0.63 mm, and 1.20 ± 0.65 mm for the SSE/Col group, the buffer control group, and the surgical control group, respectively. The difference in alveolar bone regeneration between the SSE/Col group and the surgical control group was significantly different (P <0.01). Root resorption was often observed, but no ankylosis was present. Conclusion: Wound conditioning with safflower seed extracts may contribute to bone formation but appears to have unpredictable potential for stimulating periodontal regeneration including new cementum.